Comparative transcriptome analysis of bone marrow resident versus culture-expanded mouse mesenchymal stem/stromal cells.

BACKGROUND AIMS: Mesenchymal stem/stromal cells (MSCs) are defined as culture-expanded populations, and although these cells recapitulate many properties of bone marrow (BM) resident skeletal stem/progenitor cells, few studies have directly compared these populations to evaluate how culture adaptation and expansion impact critical quality attributes. METHODS: We analyzed by RNA sequencing Lin-SCA1+ MSCs enriched from BM by immunodepletion (ID) and after subsequent culture expansion (Ex) and Lin-LEPR+ MSCs sorted (S) directly from BM. Pairwise comparisons were used to identify differentially expressed genes (DEGs) between populations, and gene set enrichment analysis was employed to identify biological pathways/processes unique to each population. K-means cluster analysis resolved isolation status-dependent changes in transcription in pseudotime. RESULTS: Hierarchical clustering segregated populations by isolation process, and principal component analysis identified transcripts related to vasculature development, ossification and inflammatory/cytokine signaling as key drivers of population variance. Pairwise comparisons identified 3849 DEGs in ID versus S BM-MSCs mapping to Gene Ontology (GO) terms related to immune and metabolic processes and 334 DEGs in Ex versus ID BM-MSCs mapping to GO terms related to tissue development, cell growth and replication and organelle organization. K-means cluster analysis revealed significant differences in transcripts encoding stemness and differentiation markers, extracellular matrix structural constituents and remodeling enzymes and paracrine-acting factors between populations. CONCLUSIONS: These comparative analyses reveal significant differences in gene expression signatures between BM resident and culture-expanded MSCs, thereby providing new insight into how culture adaptation/expansion endows the latter with unique quality attributes.

Transcriptional Targets of TWIST1 in Human Mesenchymal Stem/Stromal Cells Mechanistically Link Stem/Progenitor and Paracrine Functions.

Despite extensive clinical testing, mesenchymal stem/stromal cell (MSC)-based therapies continue to underperform with respect to efficacy, which reflects the paucity of biomarkers that predict potency prior to patient administration. Previously, we reported that TWIST1 predicts inter-donor differences in MSC quality attributes that confer potency. To define the full spectrum of TWIST1 activity in MSCs, the present work employed integrated omics-based profiling to identify a high-confidence set of TWIST1 targets, which mapped to cellular processes related to ECM structure/organization, skeletal and circulatory system development, interferon gamma signaling, and inflammation. These targets are implicated in contributing to both stem/progenitor and paracrine activities of MSCs indicating these processes are linked mechanistically in a TWIST1-dependent manner. Targets implicated in extracellular matrix dynamics further implicate TWIST1 in modulating cellular responses to niche remodeling. Novel TWIST1-regulated genes identified herein may be prioritized for future mechanistic and functional studies.

Mesenchymal stem/stromal cells from a transplanted, asymptomatic patient with Fanconi anemia exhibit an aging-like phenotype and dysregulated expression of genes implicated in hematopoiesis and myelodysplasia.

BACKGROUND AIMS: Fanconi anemia (FA) is an inherited bone marrow failure syndrome caused by defects in the repair of DNA inter-strand crosslinks and manifests as aplastic anemia, myelodysplastic syndrome and acute myeloid leukemia. FA also causes defects in mesenchymal stromal cell (MSC) function, but how different FA gene mutations alter function remains understudied. METHODS: We compared the growth, differentiation and transcript profile of a single MSC isolate from an asymptomatic patient with FA with a FANCG nonsense mutation who underwent hematopoietic stem cell transplantation 10 years prior to that from a representative healthy donor (HD). RESULTS: We show that FANCG-/- MSCs exhibit rapid onset of growth cessation, skewed bi-lineage differentiation in favor of adipogenesis and increased cellular oxidate stress consistent with an aging-like phenotype. Transcript profiling identified pathways related to cell growth, senescence, cellular stress responses and DNA replication/repair as over-represented in FANCG-/- MSC, and real-time polymerase chain reaction confirmed these MSCs expressed reduced levels of transcripts implicated in cell growth (TWIST1, FGFR2v7-8) and osteogenesis (TWIST1, RUNX2) and increased levels of transcripts regulating adipogenesis (GPR116) and insulin signaling. They also expressed reduced levels of mRNAs implicated in HSC self-maintenance and homing (KITLG, HGF, GDNF, PGF, CFB, IL-1B and CSF1) and elevated levels of those implicated in myelodysplasia (IL-6, GDF15). CONCLUSIONS: Together, these findings demonstrate how inactivation of FANCG impacts MSC behavior, which parallels observed defects in osteogenesis, HSC depletion and leukemic blast formation seen in patients with FA.

Basal p53 expression is indispensable for mesenchymal stem cell integrity.

Marrow-resident mesenchymal stem cells (MSCs) serve as a functional component of the perivascular niche that regulates hematopoiesis. They also represent the main source of bone formed in adult bone marrow, and their bifurcation to osteoblast and adipocyte lineages plays a key role in skeletal homeostasis and aging. Although the tumor suppressor p53 also functions in bone organogenesis, homeostasis, and neoplasia, its role in MSCs remains poorly described. Herein, we examined the normal physiological role of p53 in primary MSCs cultured under physiologic oxygen levels. Using knockout mice and gene silencing we show that p53 inactivation downregulates expression of TWIST2, which normally restrains cellular differentiation to maintain wild-type MSCs in a multipotent state, depletes mitochondrial reactive oxygen species (ROS) levels, and suppresses ROS generation and PPARG gene and protein induction in response to adipogenic stimuli. Mechanistically, this loss of adipogenic potential skews MSCs toward an osteogenic fate, which is further potentiated by TWIST2 downregulation, resulting in highly augmented osteogenic differentiation. We also show that p53-/- MSCs are defective in supporting hematopoiesis as measured in standard colony assays because of decreased secretion of various cytokines including CXCL12 and CSF1. Lastly, we show that transient exposure of wild-type MSCs to 21% oxygen upregulates p53 protein expression, resulting in increased mitochondrial ROS production and enhanced adipogenic differentiation at the expense of osteogenesis, and that treatment of cells with FGF2 mitigates these effects by inducing TWIST2. Together, these findings indicate that basal p53 levels are necessary to maintain MSC bi-potency, and oxygen-induced increases in p53 expression modulate cell fate and survival decisions. Because of the critical function of basal p53 in MSCs, our findings question the use of p53 null cell lines as MSC surrogates, and also implicate dysfunctional MSC responses in the pathophysiology of p53-related skeletal disorders.

Mesenchymal Stem Cells: The Moniker Fits the Science.

Mesenchymal stem cells (MSCs) have gained widespread use in regenerative medicine due to their demonstrated efficacy in a broad range of experimental animal models of disease and their excellent safety profile in human clinical trials. Outcomes from these studies suggest that MSCs achieve therapeutic effects in vivo in nonhomologous applications predominantly by paracrine action. This paracrine-centric viewpoint has become widely entrenched in the field, and has spurred a campaign to rename MSCs as "medicinal signaling cells" to better reflect this mode of action. In this Commentary, we argue that the paracrine-centric viewpoint and proposed name change ignores a wealth of old and new data that unequivocally demonstrate the stem cell nature of MSCs, and also overlooks a large effort to exploit homologous applications of MSCs in human clinical trials. Furthermore, we offer evidence that a stem cell-centric viewpoint of MSCs provides a comprehensive understanding of MSC biology that encompasses their paracrine activity, and provides a better foundation to develop metrics that quantify the biological potency of MSC batches for both homologous and nonhomologous clinical applications. Stem Cells 2018;36:7-10.

IP6K1 Reduces Mesenchymal Stem/Stromal Cell Fitness and Potentiates High Fat Diet-Induced Skeletal Involution.

Mesenchymal stem/stromal cells (MSCs) are the predominant source of bone and adipose tissue in adult bone marrow and play a critical role in skeletal homeostasis. Age-induced changes in bone marrow favor adipogenesis over osteogenesis leading to skeletal involution and increased marrow adiposity so pathways that prevent MSC aging are potential therapeutic targets for treating age-related bone diseases. Here, we show that inositol hexakisphosphate kinase 1 (Ip6k1) deletion in mice increases MSC yields from marrow and enhances cell growth and survival ex vivo. In response to the appropriate stimuli, Ip6k1-/- versus Ip6k1+/+ MSCs also exhibit enhanced osteogenesis and hematopoiesis-supporting activity and reduced adipogenic differentiation. Mechanistic-based studies revealed that Ip6k1-/- MSCs express higher MDM2 and lower p53 protein levels resulting in lower intrinsic mitochondrial reactive oxygen species (ROS) levels as compared to Ip6k1+/+ MSCs, but both populations upregulate mitochondrial ROS to similar extents in response to oxygen-induced stress. Finally, we show that mice fed a high fat diet exhibit reduced trabecular bone volume, and that pharmacological inhibition of IP6K1 using a pan-IP6K inhibitor largely reversed this phenotype while increasing MSC yields from bone marrow. Together, these findings reveal an important role for IP6K1 in regulating MSC fitness and differentiation fate. Unlike therapeutic interventions that target peroxisome proliferator-activated receptor gamma and leptin receptor activity, which yield detrimental side effects including increased fracture risk and altered feeding behavior, respectively, inhibition of IP6K1 maintains insulin sensitivity and prevents obesity while preserving bone integrity. Therefore, IP6K1 inhibitors may represent more effective insulin sensitizers due to their bone sparing properties. Stem Cells 2017;35:1973-1983.

Regulation of chronic inflammatory and immune processes by extracellular vesicles.

Almost all cell types release extracellular vesicles (EVs), which are derived either from multivesicular bodies or from the plasma membrane. EVs contain a subset of proteins, lipids, and nucleic acids from the cell from which they are derived. EV factors, particularly small RNAs such as miRNAs, likely play important roles in cell-to-cell communication both locally and systemically. Most of the functions associated with EVs are in the regulation of immune responses to pathogens and cancer, as well as in regulating autoimmunity. This Review will focus on the different modes of immune regulation, both direct and indirect, by EVs. The therapeutic utility of EVs for the regulation of immune responses will also be discussed.